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Nature Communications Apr 2022One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of...
One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.
Topics: Algorithms; DNA Primers; Likelihood Functions; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 35410464
DOI: 10.1038/s41467-022-29500-4 -
Genomics Jan 2021The ΔΔct method estimates fold change in gene expression data from RT-PCR assay. The ΔΔct estimate aggregates replicates using mean and standard deviation (sd) and...
The ΔΔct method estimates fold change in gene expression data from RT-PCR assay. The ΔΔct estimate aggregates replicates using mean and standard deviation (sd) and is not robust to outliers which are in practice often removed before the non-outlying replicates are aggregated. The alternative of using robust statistics such as median and median absolute deviation (MAD) to aggregate the replicates is not done in practice perhaps because the distribution of a robust ΔΔct estimate based on median and MAD is not straightforward to deduce. We introduce a robust ΔΔct estimate and deduce an approximate distribution for it. Simulations show that when data has outliers, the robust ΔΔct estimate compared to the non-robust ΔΔct estimate leads to significantly reduced confidence interval length and a coverage close to the nominal coverage. The analysis of an RT-PCR data from a Novartis clinical trial demonstrates benefit of a robust ΔΔct estimate.
Topics: Algorithms; Biomarkers, Tumor; Clinical Trials as Topic; Gene Expression Profiling; Humans; Real-Time Polymerase Chain Reaction; Reference Standards
PubMed: 33309766
DOI: 10.1016/j.ygeno.2020.12.009 -
Biosensors Dec 2022Pathogenic bacteria are the leading causes of food-borne and water-borne infections, and one of the most serious public threats. Traditional bacterial detection... (Review)
Review
Pathogenic bacteria are the leading causes of food-borne and water-borne infections, and one of the most serious public threats. Traditional bacterial detection techniques, including plate culture, polymerase chain reaction, and enzyme-linked immunosorbent assay are time-consuming, while hindering precise therapy initiation. Thus, rapid detection of bacteria is of vital clinical importance in reducing the misuse of antibiotics. Among the most recently developed methods, the label-free optical approach is one of the most promising methods that is able to address this challenge due to its rapidity, simplicity, and relatively low-cost. This paper reviews optical methods such as surface-enhanced Raman scattering spectroscopy, surface plasmon resonance, and dark-field microscopic imaging techniques for the rapid detection of pathogenic bacteria in a label-free manner. The advantages and disadvantages of these label-free technologies for bacterial detection are summarized in order to promote their application for rapid bacterial detection in source-limited environments and for drug resistance assessments.
Topics: Bacteria; Surface Plasmon Resonance; Polymerase Chain Reaction; Enzyme-Linked Immunosorbent Assay; Spectrum Analysis, Raman
PubMed: 36551138
DOI: 10.3390/bios12121171 -
Molecules (Basel, Switzerland) Dec 2022Polymerase Chain Reaction (PCR) is one of the most common technologies used to produce millions of copies of targeted nucleic acid in vitro and has become an... (Review)
Review
Polymerase Chain Reaction (PCR) is one of the most common technologies used to produce millions of copies of targeted nucleic acid in vitro and has become an indispensable technique in molecular biology. However, it suffers from low efficiency and specificity problems, false positive results, and so on. Although many conditions can be optimized to increase PCR yield, such as the magnesium ion concentration, the DNA polymerases, the number of cycles, and so on, they are not all-purpose and the optimization can be case dependent. Nano-sized materials offer a possible solution to improve both the quality and productivity of PCR. In the last two decades, nanoparticles (NPs) have attracted significant attention and gradually penetrated the field of life sciences because of their unique chemical and physical properties, such as their large surface area and small size effect, which have greatly promoted developments in life science and technology. Additionally, PCR technology assisted by NPs (NanoPCR) such as gold NPs (Au NPs), quantum dots (QDs), and carbon nanotubes (CNTs), etc., have been developed to significantly improve the specificity, efficiency, and sensitivity of PCR and to accelerate the PCR reaction process. This review discusses the roles of different types of NPs used to enhance PCR and summarizes their possible mechanisms.
Topics: Metal Nanoparticles; Nanotubes, Carbon; Nanostructures; Polymerase Chain Reaction; Quantum Dots; Gold
PubMed: 36557991
DOI: 10.3390/molecules27248854 -
Biosensors Jan 2024Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease... (Review)
Review
Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease mechanism. Moreover, it promotes the basic and clinical research in many fields at a single-cell resolution. A digital polymerase chain reaction (dPCR) is an absolute quantitative analysis technology with high sensitivity and precision for DNA/RNA or protein. With the development of microfluidic technology, digital PCR has been used to achieve absolute quantification of single-cell gene expression and single-cell proteins. For single-cell specific-gene or -protein detection, digital PCR has shown great advantages. So, this review will introduce the significance and process of single-cell analysis, including single-cell isolation, single-cell lysis, and single-cell detection methods, mainly focusing on the microfluidic single-cell digital PCR technology and its biological application at a single-cell level. The challenges and opportunities for the development of single-cell digital PCR are also discussed.
Topics: Polymerase Chain Reaction; Microfluidics; DNA; RNA; Single-Cell Analysis
PubMed: 38391982
DOI: 10.3390/bios14020064 -
Mikrochimica Acta Jun 2023Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however,...
Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however, is limited by the stability of droplets against thermal cycling. While the use of fluorinated oil or supplementation of surfactant could improve the stability of droplets, this process has also greatly increased the cost of ddPCR and limited post-PCR analysis. Here, we report a novel method known as gel capsule-based digital PCR (gc-dPCR) which includes a method to prepare hydrogel capsules encapsulating the PCR reaction mix, conducting PCR reaction, and readout by either quantitative PCR (qPCR) system or fluorescence microplate reader. We have compared the developed method to vortex ddPCR. Our approach results in higher fluorescence intensity compared to ddPCR suggesting higher sensitivity of the system. As hydrogel capsules are more stable than droplets in fluorinated oil throughout thermal cycling, all partitions can be quantified, thus preventing loss of information from low-concentration samples. The new approach should extend to all droplet-based PCR methods. It has greatly improved ddPCR by increasing droplets stability and sensitivity, and reducing the cost of ddPCR, which help to remove the barrier of ddPCR in settings with limited resources.
Topics: Hydrogels; Capsules; Polymerase Chain Reaction; Nucleic Acids
PubMed: 37261544
DOI: 10.1007/s00604-023-05827-7 -
ACS Applied Materials & Interfaces Jul 2022A highly precise and sensitive technology that enables DNA amplification/detection from minimal amounts of nucleic acid is expected to find applicability in genetic...
A highly precise and sensitive technology that enables DNA amplification/detection from minimal amounts of nucleic acid is expected to find applicability in genetic testing involving small amounts of samples. The use of a free enzyme in conventional DNA amplification techniques, such as the polymerase chain reaction (PCR), frequently causes side reactions (i.e., nonspecific DNA amplification) when ≤10 substrate DNA molecules are present, thereby preventing selective amplification of the target DNA. To address this issue, we have developed a novel DNA amplification system, mesoporous silica-enhanced PCR (MSE-PCR), which involves the immobilization of a thermostable DNA polymerase from (KOD DNA polymerase) into highly ordered nanopores of the mesoporous silica to control the reaction environment around the enzyme. In the MSE-PCR system using immobilized KOD DNA polymerase, such nonspecific DNA amplification was remarkably inhibited under the same conditions. Furthermore, the optimization of mesoporous silica pore sizes enabled selective and efficient DNA amplification from DNA substrates at the single-molecule level, i.e., one ten-thousandth of the amount of substrate DNA required for a DNA amplification reaction using a free enzyme. The results obtained in this study have shown that the nanopores of mesoporous silica can inhibit nonspecific reactions in DNA amplification, thereby considerably improving the specificity and sensitivity of the DNA polymerase reaction.
Topics: DNA; DNA-Directed DNA Polymerase; Enzymes, Immobilized; Immobilized Nucleic Acids; Polymerase Chain Reaction; Silicon Dioxide
PubMed: 35700238
DOI: 10.1021/acsami.2c01992 -
International Journal of Environmental... Apr 2022Microbial water quality is of vital importance for human, animal, and environmental health. Notably, pathogenically contaminated water can result in serious health... (Review)
Review
Microbial water quality is of vital importance for human, animal, and environmental health. Notably, pathogenically contaminated water can result in serious health problems, such as waterborne outbreaks, which have caused huge economic and social losses. In this context, the prompt detection of microbial contamination becomes essential to enable early warning and timely reaction with proper interventions. Recently, molecular diagnostics have been increasingly employed for the rapid and robust assessment of microbial water quality implicated by various microbial pollutants, e.g., waterborne pathogens and antibiotic-resistance genes (ARGs), imposing the most critical health threats to humans and the environment. Continuous technological advances have led to constant improvements and expansions of molecular methods, such as conventional end-point PCR, DNA microarray, real-time quantitative PCR (qPCR), multiplex qPCR (mqPCR), loop-mediated isothermal amplification (LAMP), digital droplet PCR (ddPCR), and high-throughput next-generation DNA sequencing (HT-NGS). These state-of-the-art molecular approaches largely facilitate the surveillance of microbial water quality in diverse aquatic systems and wastewater. This review provides an up-to-date overview of the advancement of the key molecular tools frequently employed for microbial water quality assessment, with future perspectives on their applications.
Topics: Drug Resistance, Microbial; Multiplex Polymerase Chain Reaction; Pathology, Molecular; Real-Time Polymerase Chain Reaction; Water Quality
PubMed: 35564522
DOI: 10.3390/ijerph19095128 -
Genes Mar 2024The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The... (Review)
Review
The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.
Topics: Humans; Polymerase Chain Reaction; Forensic Sciences; DNA Fingerprinting; DNA; Forensic Genetics
PubMed: 38674373
DOI: 10.3390/genes15040438 -
Molecular and Cellular Biochemistry Jul 2023Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore... (Review)
Review
Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore methods to detect gene mutations, which may help in disease diagnosis, medication guidance, and so on. Currently, the detection methods, such as whole-genome sequencing and polymerase chain reaction, have some limitations in terms of cost and sensitivity. Ligase (an enzyme) can recognize base mismatch as a commonly used tool in genetic engineering. Therefore, the ligase-related nucleic acid amplification technology for detecting gene mutations has become a research hotspot. In this study, the main techniques explored for detecting gene mutations included the ligase detection reaction, ligase chain reaction, rolling circle amplification reaction, enzyme-assisted polymerase chain reaction, and loop-mediated isothermal amplification reaction. This review aimed to analyze the aforementioned techniques and mainly present their advantages and disadvantages, sensitivity, specificity, cost, detection time, applications, and so on. The findings may help develop sufficient grounds for further studies on detecting gene mutations.
Topics: Ligases; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Mutation; Technology; Nucleic Acids
PubMed: 36441353
DOI: 10.1007/s11010-022-04615-w